PROJECT SUMMARY. Fibrosis affects almost every tissue in the body and is the pathological outcome of chronic inflammation or misregulated of wound healing that leads to tissue stiffening and ultimately loss of organ function with lethal consequences. For example, idiopathic pulmonary fibrosis (IPF), whose cause is unknown, is untreatable and approximately two-thirds of patients die within 5 years, where approximately 50,000 new cases are diagnosed annually. A significant fundamental and clinical need exists to understand the pathobiology of fibrosis, particularly IPF. While inflammation is thought to be key in the initiation of IPF, chronic inflammation is not observed, and defects in the wound healing response of lung fibroblasts and epithelial cells are thought to drive progression. Further, the composition and rigidity of the extracellular matrix (ECM) have been recognized as drivers, not just consequences, of the disease. However, human IPF often is caught late in development, and current animal and cell culture models provide insight but do not capture the irreversibility or complexity, respectively, of the human disease, making it currently intractable. This proposal is based on the belief that two main barriers prevent advancement toward a cure for IPF: need for i) a relevant human model system for hypothesis testing and drug screening and ii) tools for examining dynamic interactions between fibroblast, epithelial cells, and the microenvironment. We hypothesize that a human co-culture model with niche cells and temporally controlled ECM can recapitulate key aspects of human IPF and can be used to identify novel therapeutic targets for modulating fibroblast activities to mitigate progression. To test this, we propose to create a model lung interstitium that mimics the dynamic and heterogeneous structure and composition of the native fibroblast microenvironment from healthy to fibrotic tissue, allowing light-triggered stiffening and increasing collagen content to probe fibroblast response to progression. This synthetic interstitium will be co-cultured with a model lung epithelium that enables triggered injury and addition of inflammatory factors to examine both fibrosis onset and progression. Innovative tools, including new fluorescent reporter systems and live cell imaging, will be used to monitor cell activation and response in these dynamic microenvironments in real-time. Proteomic and next generation sequencing techniques will be used for validation of the model system in comparison to animal and clinical data and to identify new therapeutic targets for disease treatment. This transformative work will generate a comprehensive culture model for testing previously intractable hypotheses and for drug screening toward halting fibrosis progression.